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. 2012 Mar 20;10(3):e1001286. doi: 10.1371/journal.pbio.1001286

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Xu et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A–E) xNorrin inhibits Activin-B-mRNA-induced mesoderm formation. A wild-type (WT) embryo at a neurula stage (A). Wild-type animal caps with elongation (5% of the caps showed elongation, n = 60) (B). Elongated animal caps induced by Activin-B mRNA (25 pg) injection (82% of the injected caps showed elongation, n = 55) (C). Animal cap elongation was blocked in animal caps injected with Activin-B (25 pg) and xNorrin (200 pg) mRNAs (10% of the co-injected caps showed elongation, n = 58) (D). The Activin-B-mRNA-induced expression of mesoderm markers (Wnt8, Xbra, m-actin, and MyoD) was inhibited by xNorrin (E). RNAs were injected into the animal pole of one-cell embryos, and animal caps were cut around stage 8 and cultured in 1× MMR until the sibling embryos reached neurula stage. (F and G) xNorrin inhibits BMP4 signaling. xNorrin mRNA (500 pg), like ΔBMPR mRNA (200 pg) and Chordin mRNA (100 pg), inhibited BRE-Luc reporter activity in Xenopus embryos (F). xNorrin mRNA (500 pg) inhibited BMP4-induced Smad1 phosphorylation in animal caps (G). P-Smad1, phosphorylated Smad1; T-Smad1, total Smad1; WE, whole embryo. (H) BMP4 inhibited xNorrin-induced otx2, Xpax6, and NCAM RNA expression in animal caps of stage 15 embryos. –RT, no reverse transcription; WE, whole embryo; WT, wild-type animal caps. (I) xNorrin interacts with BMP4. BMP4-Flag and xNorrin-Myc mRNAs or xNorrin-Flag and BMP4-Myc mRNAs were injected into adjacent cells of four-cell embryos. FLAG-tagged proteins were immunoprecipitated (IP) from later gastrula embryos with a FLAG antibody. The proteins were PAGE separated and immunoblotted (IB) with an anti-c-Myc antibody. Arrowheads indicate xNorrin-Myc (top) or BMP4-Myc (bottom). L.C., IgG light chain.